Isolating DNA from plasma using a column.
DNA concentration on nanodrop is 5ng/ul.
Unable to see any band in gel after 3-5 ul load
no result in PCR amplification?
what can be the reason?
Seems like your DNA isolation has failed. Concentration 5 ng/μl is almost within Nanodrop's systematic error, so this estimate is not reliable and your samples probably contain only a trace amounts of DNA.
Try to optimize your extraction protocol. For example, increase of the exposure time of the column with binding buffer may help.
Your dna is probably below visual detection even if it were a single size band on the gel but plasma dna is randonly degraded and small so is spread over a range of sizes on the gel so no one size will be visible. Seeing dna is not very important if the next step of your research is a pcr amplification then go ahead and amplify and it will then produce visible bands
Your DNA is probably below visual detection even if it were a single size band on the gel but plasma DNA is randonly degraded and small so is spread over a range of sizes on the gel so no one size will be visible. Seeing dna is not very important if the next step of your research is a pcr amplification then go ahead and amplify and it will then produce visible bands
Alexandr Pozharskiy Thank your for the interest.
But generally the DNA is very less in blood plasma. So I am not sure whether DNA extraction failed or amplification...
Paul Rutland Ramy Essa El-Ansary Thank you so much for the answer. I tried for PCR amplification but it failed. So can you put some light for any improvement in PCR amplification using plasma DNA.
You may be looking for a minority dna.....viral or fetal or tumour dna circulating in plasma where almost all of the isolated dna is host normal degraded dna so the amount of your target dna is very small. In this case you can run a nested pcr. So if your primers are more than about 300bp apart you can first try amplifying a smaller target .
A very good way of detecting small amounts of dna is nested prc.
If your primers can be represented by bases 1-20 and 200-180 (for instance) then get 2 primers made that are 21-40 and 180-160. For nested pcr the inner primer set are amplifying an enriched target so you can be quite relaxed about primer desigm,
So amplify your dna 30 cycles with the outer set of primers ( which will appear not to amplify). The dilute the pcr product 1:100 and re amplify 1ul of diluted first amplimer 20, 25 and 30 cycles using the inner primers 21-40 and 180-160 and you should have a good amplification. of size 160 bp in this example If you get a high size smear of amplimer than try running fewer second round cycles
Paul Rutland thank you very much...
Actually, I have to amplify first approximately 300 bp then restriction digestion to check the polymorphism. so designing new primers with smaller is not possible. Or there is other way also?
You can still use nested or hemi nested pcr to get a visible product. If the polymorphism is near the middle of the sequence 50-250 bases then you can still use fully nested pcr. If the polymorphism is near to one end 21-40 or 280-260 then we use half nested pcr. Run the outer primer set. Dilute the pcr product and then if the change is near the start then use primers 1-20 and 280-260 but if the change is at the 260-280 region then use primers 21-40 and 300-280.
It is possible to design nested primers/primer and this is much more sensitive than re amplifying a pcr product with the same primers or just running more cycles of the first round pcr. If the problem is comparing sizes of restriction fragments with previous research then you just need to nest the pcr and add the length that the inner primers have not amplified to the RE fragment sizes.
Have you tested that your primers do work on a control dna sample. It is possible that your dna is ok but that your primers are degraded or that you are annealing them at too high a temperature and the pcr is simply not working or even that you have not purified very much dna with your column isolation
- Badges
- Science topic
- Similar topics
- Gels